The prokaryotic bacteria and
archaea exhibit an astonishing metabolic
diversity, which far exceeds that of animals,
plants, fungi and other higher organisms. The prokaryotes
literally keep our biological world turning by recycling
all the mineral elements necessary for life support.

Two famous microbiologists
pioneered the study of these processes: Sergius Winogradsky
(1856-1953) and Martinus Willem Beijerinck
(1851-1931). In contrast to the pure culture studies of
other pioneer microbiologists such as Louis Pasteur and
Robert Koch, these workers studied the relationships
between different types of microorganisms in mixed
communities.

A simple laboratory demonstration - the Winogradsky
column - illustrates how different
microorganisms perform their interdependent roles: the
activities of one organism enable another to grow, and
vice-versa. These columns are complete, self-contained
recycling systems, driven only by energy from light!

The columns (Figure
A) are easy to set up with a glass or
perspex tube, about 30 cm tall and 5 cm diameter. Mud
from the bottom of a lake or river is supplemented with
cellulose (e.g. newspaper), sodium sulphate and calcium
carbonate, then added to the lower one-third of the tube.
The rest of the tube is filled with water from the lake
or river, and the tube is capped and placed near a window
with supplementary strip lights.

All the organisms are present initially in
low numbers, but when the tubes are incubated for 2 to 3
months the different types of microorganism proliferate
and occupy distinct zones where the environmental
conditions favour their specific activities.

We shall return to the
'true' columns later, but first we will use an idealised
column shown below, and list some of the main activities
that occur.

1. The large amount of
cellulose added initially promotes rapid microbial growth
which soon depletes the oxygen in the sediment and in the
water column. Only the very top of the column remains
aerated because oxygen diffuses very slowly through
water.

2. The only
organisms that can grow in anaerobic conditions are those
that ferment organic matter and those that perform
anaerobic respiration. Fermentation is a
process in which organic compounds are degraded
incompletely; for example, yeasts ferment sugars to
alcohol. Anaerobic respiration is a
process in which organic substrates are degraded
completely to CO2, but using a substance other
than oxygen as the terminal electron acceptor. Some
bacteria respire by using nitrate or sulphate ions, in
the same way as we use oxygen as the terminal electron
acceptor during respiration.

3. Some cellulose-degrading
Clostridium species
start to grow when the oxygen is depleted in the
sediment. All Clostridium species are strictly
anaerobic because their vegetative cells are killed by
exposure to oxygen, but they can survive as spores in
aerobic conditions. They degrade the cellulose to glucose
and then ferment the glucose to gain energy, producing a
range of simple organic compounds (ethanol, acetic acid,
succinic acid, etc.) as the fermentation end products.

Figure B. This tube was filled with sterile
nutrient medium containing sulphate, an organic acid and
an iron nail. It was inoculated with a pure culture of Desulfovibrio,
and after 10 days the contents turned black. Desulfovibrio
has small, comma-shaped cells. Figure C. A species of Thiocapsa (purple
sulphur bacterium) from the Winogradsky column
(number 2) shown in Fig.
A at the top of this
page. Viewed by phase contrast microscopy. The purple
colour can be seen where the cells are very dense.

4. The sulphur-reducing bacteria (Fig. B) such as Desulfovibrio
can utilise these fermentation products by anaerobic
respiration, using either sulphate or other
partly oxidised forms of sulphur (e.g. thiosulphate) as
the terminal electron acceptor, generating large amounts
of H2S by this process.[In our own aerobic
respiration we use O2 and reduce it to H2O].
The H2S will react with any iron in the
sediment, producing black ferrous sulphide. This is why
lake sediments (and our household drains) are frequently
black. However, some of the H2S diffuses
upwards into the water column, where it is utilised by
other organisms.

5. The diffusion of H2S from the
sediment into the water column enables anaerobic
photosynthetic bacteria to grow. They are seen
usually as two narrow, brightly coloured bands
immediately above the sediment - a zone of green
sulphur bacteria then a zone of purple
sulphur bacteria(Fig.
C).

The green and purple
sulphur bacteria gain energy from light reactions and
produce their cellular materials from CO2 in
much the same way as plants do. However, there is one
essential difference: they do not generate oxygen during
photosynthesis because they do not use water as the
reductant; instead they use H2S. The following
simplified equations show the parallel.

The purple sulphur
bacteria typically have large cells and they deposit
sulphur granules inside the cells. The organism shown
here (Fig. C) is a species of Thiocapsa. The
green sulphur bacteria have smaller cells and typically
deposit sulphur externally.

The sulphur (or sulphate
formed from it) produced by the photosynthetic bacteria
returns to the sediment where it can be recycled by Desulfovibrio
- part of the sulphur cycle in natural
waters.

6. Most of the water column above the photosynthetic
bacteria is coloured bright red by a large population of purple
non-sulphur bacteria. These include species of Rhodopseudomonas,
Rhodospirillum and Rhodomicrobium. A
mixed culture of them is shown in the bottle in Figure
E (below).

These
bacteria grow in anaerobic conditions, gaining their
energy from light reactions but using organic acids as
their carbon source for cellular synthesis. So they are
termed photoheterotrophs. The organic
acids that they use are the fermentation products of
other anaerobic bacteria (e.g. Clostridium
species), but the purple non-sulphur bacteria are
intolerant of high H2S concentrations, so they
occur above the zone where the green and purple sulphur
bacteria are found.

The columns shown at the
top of this page (Fig. A) have passed the stage where
these organisms are common because the water columns
became oxygenated by cyanobacteria (Fig.
D).

7. Many microorganisms
can grow in the oxygenated zone at the top of the water
column, but three distinctive types are of special
interest.

(i) Any H2S
that diffuses into the aerobic zone can be oxidised to
sulphate by the sulphur-oxidising bacteria.
These bacteria gain energy from oxidation of H2S,
and they synthesize their own organic matter from CO2.
So they are termed chemosynthetic
organisms, or chemoautotrophs. Similar
types of organism occur in soils, gaining energy from the
oxidation of ammonium to nitrate, which then leaches from
the soil and can accumulate in water supplies.

(ii) Photosynthetic cyanobacteria
can grow in the upper zones. These are the only bacteria
that have oxygen-evolving photosynthesis
like that of plants. In fact, there is very strong
evidence that the chloroplasts of plants originated as
cyanobacteria (or the ancestors of present-day
cyanobacteria) that lived as symbionts inside the cells
of a primitive eukaryote. Similarly, there is equally
strong evidence that the mitochondria of present-day
eukaryotes were derived from purple bacteria.

Once the cyanobacteria
start to grow they can oxygenate much of the water. This
happened in column 2 (Fig. A) at the top
of this page - the whole water column was dominated by a
mass of cyanobacteria composed of spiral filaments (see
Fig. D).

(iii) The top of the
water column can contain large populations of sheathed
bacteria (se Column 1, Fig. A). These are
aerobic organisms which use organic substrates, but are
unusual because as the bacterial cells divide they
synthesize a rigid tubular sheath from which individual
cells can escape and swim away to establish new colonies.
Many empty sheaths are seen in older colonies. They are
made of a complex mixture of protein, polysaccharide and
lipid, and are thought to protect the cells from
predation by protozoa. The sheaths also can be encrusted
with ferric hydroxide, giving a yellow or rusty
appearance to the colonies.

Narrow sheaths of the
sheathed bacteria, taken from the upper yellow-orange
zone of the Winogradsky column (labelled 1)
in Fig. A. Most of the sheaths were empty. Same
magnification as used for the cyanobacterium in Fig. D.

Summary

The
Winogradsky column is a classic demonstration of the
metabolic diversity of prokaryotes. All life on earth can
be categorised in terms of the organism's carbon and
energy source: energy can be obtained from light
reactions (phototrophs) or from chemical
oxidations (of organic or inorganic substances) (chemotrophs);
the carbon for cellular synthesis can be obtained from CO2
(autotrophs) or from preformed organic
compounds (heterotrophs). Combining
these categories, we get the four basic life strategies:
photoautotrophs (e.g. plants), chemoheterotrophs (e.g.
animals, fungi), photoheterotrophs and chemoautotrophs.
Only in the bacteria - and among the bacteria within a
single Winogradsky column - do we find all four basic
life strategies.

The Winogradsky column is
also a classic demonstration of how microorganisms occupy
highly specific microsites according to their
environmental tolerances and their carbon and energy
requirements.

And, finally, the column
enables us to see how mineral elements are cycled in
natural environments. We focused mainly on sulphur, but
there are equivalent cycles for nitrogen, carbon and
other elements.